Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na⁺K⁺ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 μM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na⁺K⁺ ATPase activity in whole cells, Na⁺K⁺ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na⁺K⁺ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na⁺K⁺ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na⁺K⁺ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na⁺K⁺ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.     

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.      

Mutants of the Clo DF13 plasmid inEscherichia coli with a decreased bacteriocinogenic activity

Summary Three Clo DF13 mutant plasmids (designated asclp03, clp05 andclp21) that show a decreased cloacin activity were isolated. The decreased cloacin activity was not due to a reduced number of Clo DF13 copies per cell. The cloacins produced by theclp03 and theclp21 mutant plasmids have a strongly decreased killing activityin vivo in comparison with the wild type cloacin and the cloacin of theclp05 mutant plasmid. Furthermore no lacunae could be observed fromclp03 orclp21 harbouring strains, while strains harbouring theclp05 plasmid showed a 50–100 times decreased frequency of lacunae. In addition theclp05 mutant showed a decreased rate of RNA synthesis inclp05 harbouringEscherichia coli minicells. No complementation between the three mutant plasmids was observed. We suggest that theclp03 andclp21 mutations are located in the gene coding for the cloacin. Since the cloacin produced by theclp05 mutant plasmid has retained all the known wild type cloacin activities, the reduced inhibition zone in the stab test is probably caused by a mutation affecting the expression of the cloacin gene. The nature of this mutation is discussed.     

Cl− channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl− channels with apical Cl− channels from jejunum and trachea

Summary Cl^– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl^– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl^– in the absence of PKA. The PKA requirement for activation of Cl^– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl^– concentrations on the properties of Cl^– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl^– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl^– concentration from 2 to 50 mm increased both channel activity (P _o) and channel conductance (g _Cl, pS). Likewise, trans PKA increased mTALH Cl^– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g _Cl. Moreover, when mTALH Cl^– channels were inactivated by reducing cis Cl^– concentrations to 50 mm, cis PKA addition did not affect P _o. These results are consistent with the view that these Cl^– channels originated from basolateral membranes of the mTALH.Cl^– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl^– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl^– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl^– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl^– channel activity by both intracellular Cl^– and by PKA differ in basolateral mTALH Cl^– channels compared to apical Cl^– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator.     

Protective effects of vitamins and selenium compounds in yeast

Antimutagens and anticarcinogens are known to play an important role in decreasing damages induced by oxidants. In this study, we investigated the genotoxic and antimutagenic potential of two selenium compounds (sodium selenite: Na(2)SeO(3); seleno-DL-methionine: C(5)H(11)NO(2)Se) and Vitamins A and E in yeast cells of Saccharomyces cerevisiae. An oxidative mutagen (hydrogen peroxide (H(2)O(2)), HP) was chosen as positive control. We determined the enzymatic activities involved in the protection against oxidative damages (catalase: CAT; superoxide dismutase: SOD; glutathione peroxidase: GPx) in the cytosolic extract of yeast cells. The results demonstrated that selenium compounds exerted both mutagenic and antimutagenic effect at different concentrations. Antimutagenesis was evident both in stationary and in logarithmic phase cells. Catalase, SOD, and GPx were significantly increased in the presence of all the compounds assayed. Vitamins A (retinol) and E (alpha-tocopherol) did not have toxic or mutagenic action.     

Integrative analysis of the mitochondrial proteome in yeast

In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.     

Analytic assessment of the various bioimpedance methods used to estimate body water

Knowledge of patient fluid distribution would beuseful clinically. Both single-frequency (SF) and impedance modelingapproaches are proposed. The high intercorrelation between body watercompartments makes determining the best approach difficult. This studywas conducted to evaluate the merits of an SF approach. Mathematical simulation was performed to determine the effect of tissue change onresistance and reactance. Dilution results were reanalyzed, andresistance and parallel reactance were used to predict the intracellular water for two groups. Results indicated that the amountof intracellular and extracellular water conduction at any SF can varywith tissue change, and reactance at any SF is affected by all tissueparameters. Modeling provided a good prediction of dilutionintracellular and extracellular water, but an SF method did not.Intracellular, extracellular, and total body water were equallypredicted at all frequencies by SF resistance and parallel reactance.Extracellular and intracellular water are best measured throughmodeling, because only at the zero and infinite frequencies are theresults sensitive only to extracellular and intracellular water. At allother frequencies there are other effects.

     

Psychotropic Drug Use in S?o Paulo,Brazil – An Epidemiological Survey

Objective

To estimate the prevalence of one month psychotropic drug use in São Paulo, Brazil, and to assess the gap treatment between the presence of mental disorders and psychotropic drug users.

Method

A probabilistic sample of non-institutionalized individuals from the general population of São Paulo (n = 2336; turnout: 84.5%) who were 15 years or older were interviewed by a trained research staff, applying the Composite International Diagnostic Interview 2.1 (CIDI WHO) (depression, anxiety-phobia, OCD\PTSD, alcoholism sections), and an inventory investigating psychotropic drug use during the 12-month and one-month periods immediately preceding the interview. Logistic models were fitted to investigate associations between psychotropic drug use as well as socio-demographic and clinical variables.

Results

The one month prevalence of psychotropic drug use in São Paulo was 5.89%, the most commonly used drugs were antidepressants (3.15%) and tranquilizers (2.67%). A higher consumption of psychotropic drugs (overall, antidepressants and tranquilizers) was observed among women (OR:2.42), older individuals (OR:1.04), individuals with higher levels of formal education (1.06), and individuals with a family (OR:2.29) or personal history of mental illness (OR:3.27). The main psychotropic drug prescribers were psychiatrists (41%), followed by general practitioners (30%); 60% of psychotropic drugs were obtained through a government-run dispensing program. Most individuals who obtained a positive diagnosis on the CIDI 2.1 during the previous month were not using psychotropic medication (85%). Among individuals with a diagnosis of moderate to severe depression, 67.5% were not on any pharmacological treatment.

Conclusion

There is a change in the type of psychotropic more often used in São Paulo, from benzodiazepines to antidepressants, this event is observed in different cultures. The prevalence of use is similar to other developing countries. Most of the patients presenting a psychiatric illness in the month prior to testing were not receiving any sort of psychiatric medication. This may be explained by a failure to identify cases in primary care, which could be improved (and access to treatment could be facilitated) if professionals received more specialized training in managing cases with mental health problems.     

Epstein-Barr Virus MicroRNA Expression Increases Aggressiveness of Solid Malignancies

The Cancer Genome Atlas (TCGA) microRNA (miRNA) initiative has revealed a pivotal role for miRNAs in cancer. Utilizing the TCGA raw data, we performed the first mapping of viral miRNA sequences within cancer and adjacent normal tissues. Results were integrated with TCGA RNA-seq to link the expression of viral miRNAs to the phenotype. Using clinical data and viral miRNA mapping results we also performed outcome analysis. Three lines of evidence lend credence to an active role of viral miRNAs in solid malignancies. First, expression of viral miRNA is consistently higher in cancerous compared to adjacent noncancerous tissues. Second, viral miRNA expression is associated with significantly worse clinical outcome among patients with early stage malignancy. These patients are also featured by increased expression of PD1/PD-L1, a pathway implicated in tumors escaping immune destruction. Finally, a particular cluster of EBV-miRNA (miR-BART2, miR-BART4, miR-BART5, miR-BART18, and miR-BART22) is associated with expression of cytokines known to inhibit host response to cancer. Quantification of specific viral miRNAs may help identify patients who are at risk of poor outcome. These patients may be candidates for novel therapeutic strategies incorporating antiviral agents and/or inhibitors of the PD-1/PD-L1 pathway.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.